Project Summary. A grand challenge of the 21st century is to gain a complete understanding of the brain's most fundamental characteristics, which include its potential for plasticity and its ability to learn from experience. Activity- dependent gene expression is central to neural plasticity, learning, and memory; however, efforts to elucidate the underlying mechanisms in the brain have remained elusive due to conceptual limitations in appreciating how our genome rapidly adapts to environmental changes and technical limitations with regard to molecular tools to interrogate this process. For example, the transcriptional programs of individual neurons are highly specific, and this is obscured by the massive diversity of cell-types in the adult brain that are also organized in a region specific manner. Therefore, a major hurdle has been the lack of tools to study dynamic gene expression programs in the brain, especially in real-time. The primary goal of this R21 proposal is to develop a novel experimental program to track nascent transcription in two cell types (neurons and astrocytes), simultaneously within a living brain. Our strong preliminary data demonstrates that we have identified orthogonal neucleoside/nucleobase-enzyme pairs that can be exploited for nascent metabolic labeling of RNA. Our specific aims are to optimize this system within cultured neuronal cells in vitro, and to transfer our optimized protocols into a living mouse brain. Successful completion of our aims is sure to empower us with tools and preliminary data to tackle RNA transcription in a living brain during learning and memory formation.